Steroids are synthesized in the CNS, exert profound modulatory effects of receptors for amino acid neurotransmitters, and may act as endogenous modulators of synaptic activity. Abnormal activation of amino acid receptors has been proposed to play a role in the etiology of a variety of psychiatric disorders such as anxiety, depression, and schizophrenia, and to contribute to neuronal damage associated with stroke, seizures, neuropathic pain, trauma, and neurodegenerative disease (such as Parkinson's and Alzheimer's). In following up our original discovery that pregnenolone sulfate (PS), an abundant neurosteroid, modulates glutamate receptors, we have identified a variety of steroids that can potentiate (by up to 10-fold) or inhibit glutamate receptor function and exacerbate or protect against NMDA-induced death of hippocampal neurons in culture. One inhibitor compound, pregnanolone hemisuccinate, works in vivo to reduce middle cerebral artery occlusion-induced degeneration of cortical and subcortical nervous tissue. A major objective of this project will be to test our working hypothesis that steroids interact with the extracellular domain of individual receptor subunits and exert their effects by regulating the balance between excitation and inhibition between CNS neurons. Toward this end, novel steroids will be synthesized and evaluated electrophysiologically (using whole-cell and two-electrode voltage clamp) to test specific structure-activity hypotheses and to evaluate the modulatory effects of steroids on native glutamate receptors in primary rat hippocampal neurons and on homomeric receptors expressed in Xenopus oocytes. Initial results that steroids can discriminate among glutamate receptor subtypes will be extended to search for more highly selective compounds. The effects of steroids on receptor chimeras and on receptors with single amino acid substitutions will be examined to identify specific amino acids essential for ligand recognition. Novel steroids will be evaluated for their ability to inhibit EAA-induced increases in cytosolic free [Ca2+] (using Fluo-3 fluorescence as an indicator) and excitotoxic neuronal death. Collectively these experiments will add to our understanding of the molecular mechanisms whereby steroids modulate EAAR function, and to the development of neuroprotective agents and cognitive enhancers.